Identification and partial characterization of new cell density-dependent nucleocytoplasmic shuttling proteins and open chromatin

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.

(A) hsSKM cells were cultured at low and high density and fractionated using Cell Signaling Technology cell fractional kit (#9038) (Cyp: Cytoplasmic, M: Membrane, NC: Nucleus and cytoskeleton).YAP1 and histone H3 were detected by western blotting using mouse anti-YAP and histone H3 antibodies, respectively.(B) Immunofluorescence of hsSKM cells at low and high density.YAP1 was stained with anti-YAP1 antibody followed by Alexa 594 secondary antibody.Nuclei were stained with Hoechst 33342.Scale: 100 ✕ 100 μm.(A) pcDNA3-ZBTB2-HA (1 μg) diluted in water with primary amine was digested with 1 μl of DNase I (1U/μl) in 10 μl of PBS for 10 min at 37℃.For same experiment, plasmid was incubated with 2.5 μl of 5mM DSP solubilized in DMSO (final concentration is 1mM) for 30 min at room temperature.The reaction was stopped by adding 0.375 μl of 1M Tris-HCl, pH8.0.Note that protein-free plasmid DNA is non-reactive to DSP.DNA was analyzed on 0.8% agarose gel and stained with SuperGreen (Biosharp).(B) Total lysate is the cells lysed with RIPA-T buffer.DSP pellet is the pellet fraction from cells that were crosslinked with DSP, lysed with RIPA-T buffer, and then centrifuged.The cells were also lysed with RIPA-T buffer containing 6M guanidine (RIPA-G) or 1% SDS.The pellets were washed with PBS several times to remove remaining detergents.Note that additions of these detergents remove cytoskeletal actin and majority of other proteins (right panel: CBB stained SDS-PAGE gel), while retaining YAP in the pellet.Since we noticed that addition of 1% SDS reduces the efficiency of the following MNase digestion, we use RIPA-G in our experiment.(kDa)

25-kDa
CBFB antibodies used for immunofluorescent microscopy.Mouse embryonic fibroblast (MEF).(C) HEK 293 cells were cultured at either low or high density for the detection of PTGES3, and hsSKM cells were cultured at either low or high density for the detection of CBFB.Both cell types were then fixed.PTGES3 was detected using immunofluorescent microscopy with a specific anti-PTGES3 antibody, followed by a secondary antibody conjugated with Alexa594.CBFB was detected using immunofluorescence with a specific anti-PEBPβ (CBFB) mouse monoclonal antibody, followed by a secondary antibody conjugated with Alexa594.Nuclei were stained with Hoechst 33342.Scale: 100 ✕ 100 μm.Fluorescent intensity on yellow line was measured by "Plot Profile" on NIH Image J using.Red line indicates the intensity of staining of a protein of interest.Blue line indicates the intensity of nuclear staining.Pearson's correlation coefficient, calculated with GraphPad Prism 9.0.0, was used to evaluate the correlation between the intensities of the blue and red channels and then plotted.Pearson's correlation coefficient ranges from -1 (representing perfect cytosolic localization) to 1 (representing perfect nuclear localization).Each data point represents a measured cell (n=5∼10).Significance was analyzed by unpaired t-test.*P<0.05,**P<0.01,***P<0.001,****P<0.0001.ns: not significant.(D) Biochemical fractionation of CBFB.The hsSKM cells cultured at low and high density were crosslinked with DSP, and the DNA-protein complex was extracted following the procedure detailed in Figure 3.The total protein quantity was standardized using western blotting against histone before centrifugation.The CBFB quantity in each fraction was determined through western blotting, and the relative band intensity was plotted.Significance was analyzed by unpaired t-test.*P<0.05(n=3).Due to the ineffectiveness of the anti-PTGES3 antibody used for immunofluorescent microscopy in western blotting after cross-linking with DSP, the fractionation of PTGES3 couldn't be tested.After MNase digestion, soluble DNA-protein complex was separated on agarose gel and the complex was extracted from the gel.Bound protein was removed by de-crosslinking and proteinase K digestion and DNA was purified using spin column.Note that purified DNA migrated around 200 bp.(A) MNase-digested DNA fragments from cells at both low and high densities were subjected to NGS, and bed files were generated using the Galaxy platform (https://usegalaxy.org).For comparative purposes, publicly available ChIP-seq data for histone H3, H3K4me3, and H3K79me2 were utilized.Sequencing reads were aligned using Bowtie2, and macs2 was employed to create the narrowPeak file.After modifying the narrowPeak file to ensure compatibility with GREAT analysis, the data was assessed via GREAT (http://great.stanford.edu/public/html/)to identify the genes associated with these genomic regions.In the graphical representation, white bars denote genomic regions linked to one or more genes, while red bars signify genomic regions not associated with any genes.Notably, a significant proportion of peaks in the histone H3 ChIP-seq data are unrelated to protein-coding genes.In contrast, MNase-digested DNA fragments from both low-and high-density cells exhibit associations with multiple genes, similar to H3K4me3 and H3K79me2.(B) Regional distribution.From left to right, Prom: Promoters, Immediate Downstream (ImDown), 5'UTRs, 3'UTRs, Exons, Introns, and intergenic region (InterGen: non-protein-coding).Notably, the majority of the peaks in histone H3 ChIP-seq are mapped to the intergenic non-protein-coding region.However, similar to H3K4me3 ChIP-seq data, approximately 13% of MNase-digested DNA fragments from both low (12.6 %) and high (13.2%) density cells are mapped to promoter region (H3K4me3: 14.3 %).
Source image data for Figure 1, Figure 3ABC, Figure S1A, Figure S2AB, Figure S5 Only the lanes outlined in red pertain to this paper.

sFigure S1 .
Figure S1.Commercial fractionation kit does not retain YAP1 in "nuclear fraction".(A) hsSKM cells were cultured at low and high density and fractionated using Cell Signaling Technology cell fractional kit (#9038) (Cyp: Cytoplasmic, M: Membrane, NC: Nucleus and cytoskeleton).YAP1 and histone H3 were detected by western blotting using mouse anti-YAP and histone H3 antibodies, respectively.(B) Immunofluorescence of hsSKM cells at low and high density.YAP1 was stained with anti-YAP1 antibody followed by Alexa 594 secondary antibody.Nuclei were stained with Hoechst 33342.Scale: 100 ✕ 100 μm.

Figure S2 .
Figure S2.Reactivity of DSP to DNA and enrichment of protein-DNA complex by washing.(A)pcDNA3-ZBTB2-HA (1 μg) diluted in water with primary amine was digested with 1 μl of DNase I (1U/μl) in 10 μl of PBS for 10 min at 37℃.For same experiment, plasmid was incubated with 2.5 μl of 5mM DSP solubilized in DMSO (final concentration is 1mM) for 30 min at room temperature.The reaction was stopped by adding 0.375 μl of 1M Tris-HCl, pH8.0.Note that protein-free plasmid DNA is non-reactive to DSP.DNA was analyzed on 0.8% agarose gel and stained with SuperGreen (Biosharp).(B) Total lysate is the cells lysed with RIPA-T buffer.DSP pellet is the pellet fraction from cells that were crosslinked with DSP, lysed with RIPA-T buffer, and then centrifuged.The cells were also lysed with RIPA-T buffer containing 6M guanidine (RIPA-G) or 1% SDS.The pellets were washed with PBS several times to remove remaining detergents.Note that additions of these detergents remove cytoskeletal actin and majority of other proteins (right panel: CBB stained SDS-PAGE gel), while retaining YAP in the pellet.Since we noticed that addition of 1% SDS reduces the efficiency of the following MNase digestion, we use RIPA-G in our experiment.

Figure S4 .
Figure S4.DSP crosslinking does not interfere staining of CBFB by immunofluorescent microscopy.hsSKM cells were fixed with DSP and CBFB was detected by immunofluorescent using anti-CBFB rabbit polyclonal antibody following by secondary antibody conjugated with Alexa594.Similar result was obtained with anti-CBFB rabbit monoclonal antibody (CST) used for ChIPseq.Nuclei were stained with Hoechst 33342.Scale: 100 ✕ 100 μm.

Figure S6 .
Figure S6.MNase-sensitive sites on chromatin and the histone modification landscape in human skeletal muscle cells.Representative integrative genomics view of MNase-sensitive sites on chromatin purified from low-and high-density cells, and the H3K4me3 density (GSM733637), the H3K79me2 density (GSM733741), and the histone H3 density (GSE213851) at the TGFB2 region in human skeletal muscle cells.Red square indicates upstream of TGFB2 which is a target of CBFB.